RESEARCH ARTICLE PRD-2 directly regulates casein kinase I and counteracts nonsense-mediated decay in the Neurospora circadian clock Christina M Kelliher1, Randy Lambreghts1, Qijun Xiang1, Christopher L Baker1,2, Jennifer J Loros3, Jay C Dunlap1* 1Department of Molecular & Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, United States; 2The Jackson Laboratory, Bar Harbor, United States; 3Department of Biochemistry & Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, United States Abstract Circadian clocks in fungi and animals are driven by a functionally conserved transcription–translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense-Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I. *For correspondence: [email protected] Introduction The Neurospora circadian oscillator is a transcription–translation feedback loop that is positively reg- Competing interests: The ulated by the White Collar Complex (WCC) transcription factors, which drive expression of the nega- authors declare that no tive arm component Frequency (FRQ). In this way the fungal core circadian oscillator shares a competing interests exist. common regulatory architecture with the mammalian core clock. In Neurospora, the circadian nega- Funding: See page 17 tive arm complex is composed of FRQ and FRQ-interacting RNA helicase (FRH), which together Received: 14 October 2020 bring casein kinase I (CKI) to promote phosphorylation of WCC on key phospho-sites to inhibit its Accepted: 08 December 2020 activity (Wang et al., 2019). FRQ is extensively regulated transcriptionally, translationally, and post- Published: 09 December 2020 translationally over the circadian day leading ultimately to its inactivation (reviewed in: Hurley et al., 2016). Reviewing editor: Detlef Indeed, in both animals and fungi, the negative arm components are regulated at the RNA and Weigel, Max Planck Institute for Developmental Biology, protein levels to maintain circadian phase and period, and many of the molecular details of this regu- Germany lation, the focus of this paper, are conserved. Negative arm components FRQ and PER are regulated by anti-sense transcription (Koike et al., 2012; Kramer et al., 2003), by thermally regulated splicing Copyright Kelliher et al. This (Colot et al., 2005; Majercak et al., 1999), and display characteristics of intrinsically disordered pro- article is distributed under the teins (Pelham et al., 2020). Another highly conserved feature of fungal, insect, and mammalian neg- terms of the Creative Commons Attribution License, which ative arm components is progressive phosphorylation leading to their inactivation (Baker et al., permits unrestricted use and 2009; Ode et al., 2017; Vanselow et al., 2006) (reviewed in: Dunlap and Loros, 2018). Taken redistribution provided that the together, FRQ, PERs, and CRYs are tightly regulated and underlying mechanisms are often con- original author and source are served between clock models despite evolutionary sequence divergence of these negative arm credited. components. Kelliher et al. eLife 2020;9:e64007. DOI: https://doi.org/10.7554/eLife.64007 1 of 22 Research article Genetics and Genomics In contrast, less is known about the mechanisms regulating expression of the other essential member of the negative arm complex, CKI, orthologs of which are highly conserved in sequence and in function across eukaryotic clocks. CKI forms a stable complex as FRQ–FRH–CKIa in Neuros- pora (Baker et al., 2009; Go¨rl et al., 2001), as PER-DOUBLETIME (DBT) in flies (Kloss et al., 2001), and as a multi-protein complex of PER-CRY-CKId in mouse (Aryal et al., 2017). Fungal CKI phos- phorylates both FRQ and WCC (He et al., 2006). Insect DBT and mammalian CKId/e are key regula- tors of the PER2 phospho-switch, differentially phosphorylating two regions that control PER2 turnover (Top et al., 2018; Zhou et al., 2015). Thus, CKI phosphorylations contribute to feedback loop closure in all species. FRQ–CKI binding strength is a key regulator of period length and an important oscillator variable first described in Neurospora (Liu et al., 2019). CKI abundance is not rhythmic in any species described to date (Go¨rl et al., 2001; Kloss et al., 2001), but preliminary evi- dence suggests that its expression levels are tightly controlled to keep the clock on time, just like FRQ/PER/CRY. In mammals, CKI knockdown or knockout significantly lengthens period (Isojima et al., 2009; Lee et al., 2009; Tsuchiya et al., 2016), and CKId levels are negatively regu- lated by m6A methylation (Fustin et al., 2018). In Neurospora, decreasing the amounts of the casein kinase I (ck-1a) transcript using a regulatable promoter leads to long period defects up to ~30 hr (Mehra et al., 2009). CKI has a conserved C-terminal domain involved in autophosphorylation and inhibition of kinase activity (Gietzen and Virshup, 1999; Guo et al., 2019). Fungal mutants lacking this CKI C-terminal inhibitory domain have hyperactive kinase activity (Querfurth et al., 2007). Across clock models, the circadian period is sensitive to CKI abundance and activity due to its impor- tance in circadian feedback loop closure. Our modern understanding of the circadian clock was founded on genetic screens and characteri- zation of mutants with circadian defects (Feldman and Hoyle, 1973; Konopka and Benzer, 1971; Ralph and Menaker, 1988). The fungal clock model Neurospora crassa has been a top producer of relevant circadian mutants due to its genetic tractability, ease of circadian readout, and functional conservation with the animal circadian clock (reviewed in: Loros, 2020). Forward genetic screens used the ras-1bd mutant background (which forms distinct bands of conidiophores once per subjec- tive night) in race tube (RT) assays to identify key players in the circadian clock (Belden et al., 2007; Feldman and Hoyle, 1973; Sargent et al., 1966). Genetic epistasis among the period genes, and in some cases, genetic mapping of mutations was also performed using N. crassa (Feldman and Hoyle, 1976; Gardner and Feldman, 1981; Morgan and Feldman, 2001). The period (prd) mutants in Neurospora are distinct from the Drosophila gene period (per) (Konopka and Benzer, 1971) and its mammalian orthologs. All but one of the extant period genes in Neurospora have been cloned, and their identities have expanded our knowledge of core-clock modifying processes. prd-4 (period-4), encoding checkpoint kinase 2 (Chk2), links the clock to cell-cycle progression (Pregueiro et al., 2006). prd-3 (period-3), encoding casein kinase II (CKII), implicated direct phosphorylation of core clock proteins as central to temperature compensation (Mehra et al., 2009). prd-1 (period-1) encodes an essential RNA-heli- case that regulates the core clock under high nutrient environments (Emerson et al., 2015). prd-6 (period-6, hereafter referred to as upf1prd-6) encodes the core UPF1 subunit of the Nonsense-Medi- ated Decay (NMD) complex (Compton, 2003), although its circadian role remains cryptic. Among the available prd genes, only prd-2 (period-2) remains uncharacterized. We have mapped the prd-2 mutation to NCU01019 using whole genome sequencing, and discov- ered its molecular identity; however, attributing its long period mutant phenotype to molecular func- tion has remained elusive (Lambreghts, 2012). Equipped with the identity of PRD-2, we then followed up on the observation that the upf1prd-6 short period phenotype is completely epistatic to the prd-2 mutant’s long period (Morgan and Feldman, 1997; Morgan and Feldman, 2001). We find that UPF1PRD-6 and PRD-2 use distinct mechanisms to play opposing roles in regulating levels of the casein kinase I transcript in Neurospora, thus rationalizing the circadian actions of the two clock mutants whose roles in the clock were not understood. PRD-2 stabilizes the ck-1a mRNA transcript, and the clock-relevant domains and biochemical evaluation of the PRD-2 protein indicate that it acts as an RNA-binding protein. We genetically rescue the long period phenotype of prd-2 mutants by expressing a hyperactive CKI allele and by titrating up ck-1a mRNA levels using a regulatable pro- moter. The endogenous ck-1a transcript has a strikingly long 3’-UTR, indicating that its mRNA could be subject to NMD during a normal circadian day. We confirm that upf1prd-6 mutants have elevated levels of ck-1a in the absence of NMD, and further rescue the short period defect of upf1prd-6 Kelliher et al. eLife 2020;9:e64007. DOI: https://doi.org/10.7554/eLife.64007 2 of 22 Research article Genetics and Genomics mutants by titrating down ck-1a mRNA levels using an inducible promoter. Taken together, a unify- ing model emerges to explain the action of diverse